Acquired drug resistance to currently available chemotherapeutic drugs is a major cause of failure of cancer treatment (Gottesman et al., 1993, Annu Rev. Biochem 62:385-427; Van Der Zee et al., 1995, Gynecologic Oncol. 58:165-178; Casazza et al., 1996, Cancer Treat. Res. 87:1-171). Many of the most common carcinomas, including breast and ovarian cancer, are initially relatively sensitive to a wide variety chemotherapy agents. However, acquired drug resistance phenotype typically occurs after months or years of exposure to chemotherapy. Determining the molecular basis of drug resistance may offer opportunities for improved diagnostic and therapeutic strategies.
Taxol is a natural product derived from the bark of Taxus brevafolio (Pacific yew). Taxol inhibits microtubule depolymerization during mitosis and results in subsequent cell death. Taxol displays a broad spectrum of tumorcidal activity including against breast, ovary and lung cancer (McGuire et al., 1996, N. Engld. J. Med. 334:1-6; and Johnson et al., 1996, J. Clin. Ocol. 14:2054-2060). While taxol is often effective in treatment of these malignancies, it is usually not curative because of eventual development of taxol resistance. Cellular resistance to taxol may include mechanisms such as enhanced expression of P-glycoprotein and alterations in tubulin structure through gene mutations in the xcex2 chain or changes in the ratio of tubulin isomers within the polymerized microtubule (Wahl et al., 1996, Nature Medicine 2:72-79; Horwitz et al., 1993, Natl. Cancer Inst. 15:55-61; Haber et al., 1995, J. Biol. Chem. 270:31269-31275; and Giannakakou et al., 1997, J. Biol. Chem. 272:17118-17125). Some tumors acquires taxol resistance through unknown mechanisms.
A gene that is overexpressed in taxol-resistant cancer cell lines has now been discovered and characterized. The gene is designated Taxol Resistance Associated Gene-3 (xe2x80x9cTRAG-3xe2x80x9d). At least two alternatively spliced forms of TRAG-3, referred to herein as TRAG-3xcex1 and TRAG-3xcex2, exist. The TRAG-3xcex1 cDNA sequence shown in FIG. 2 encodes a 110 amino acid protein. The TRAG-3xcex2 cDNA shown in FIG. 3 encodes a 127 amino acid protein (FIG. 4). TRAG-3 is expressed predominantly in chemotherapy resistant cell lines and the majority of melanoma cell lines and melanoma malignant tissues. It is minimally, if at all, expressed in normal cells and tissues. As used herein, the term TRAG-3 refers to any and all TRAG-3 gene products. The TRAG-3 gene product, i.e., the TRAG-3 polypeptide, modulates expression of several genes involved in taxol resistance displayed by mammalian cells.
Based on the discoveries, the invention features an isolated nucleic acid containing a nucleotide sequence that encodes a polypeptide that shares at least 80% sequence identity with SEQ ID NO:2 or SEQ ID NO:4. Preferably, it shares at least 90% sequence identity with SEQ ID NO:2 or SEQ ID NO:4, and more preferably, it shares at least 95% sequence identity with SEQ ID NO:2 or SEQ ID NO:4. Alternatively, the nucleotide sequence defines a DNA molecule whose complement hybridizes under stringent hybridization conditions to a DNA molecule having a sequence consisting of SEQ ID NO:1 or SEQ ID NO:3.
The invention also includes an isolated nucleic acid (antisense) that hybridizes under stringent hybridizaton conditions to a nucleic acid whose nucleotide sequence is the complement of SEQ ID NO:1 or SEQ ID NO:3.
The invention also includes an isolated nucleic acid containing a nucleotide sequence that encodes: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:2 with one or more conservative amino acid substitutions; or SEQ ID NO:4 with one or more conservative amino acid substitutions. In some embodiments, the nucleotide sequence is SEQ ID NO:1 or SEQ ID NO:3.
Preferably, the polypeptide modulates expression of at least one gene involved in taxol resistance displayed by a mammalian cell. Examples of genes involved in taxol resistance include: annexin I, interleukin 6 (IL-6), interleukin 8 (IL-8), macrophage inflammatory protein 2xcex1, and natural killer cell enhancing factor B (NKEFB).
The invention also includes a vector containing an above-described nucleic acid. In the vector, the nucleic acid can be operably linked to at least one expression control sequence. The invention also includes a cell comprising the vector.
The invention also includes a substantially pure polypeptide that has an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO:2 or SEQ ID NO:4. The invention also includes a substantially pure polypeptide whose amino acid sequence is: SEQ ID NO:2; SEQ ID NO:4, SEQ ID NO:2 with one or more conservative amino acid substitutions; or SEQ ID NO:2 with one or more conservative amino acid substitutions. Preferably, the polypeptide modulates expression of at least one gene involved in taxol resistance displayed by a mammalian cell, e.g., annexin I, IL-6, IL-8, macrophage inflammatory protein 2xcex1, and NKEFB.
The invention also includes an antibody that binds to a TRAG-3 polypeptide. Preferably, the antibody binds specifically to a TRAG-3 polypeptide, or a biologically active portion thereof. The antibody can be a monoclonal, polyclonal, or engineered antibody. The antibody is useful in detecting a TRAG-3 polypeptide, e.g., in a biological sample, or to alter the activity of TRAG-3.
The invention also includes a method for modulating expression of annexin I, IL-6, IL-8, macrophage inflammatory protein 2xcex1, or NKEFB, in a mammalian cell. The method includes introducing into the cell a vector containing a TRAG-3 coding sequence operably linked to expression control sequences.
The invention also includes a screening method for identifying a compound that inhibits expression of a TRAG-3 gene in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which express endogenous TRAG-3, (b) contacting the treatment test cell with a candidate compound, and (c) detecting a decrease in the level of TRAG-3 gene expression in the treatment test cell, compared to the level of TRAG-3 gene expression in the control test cell. The test cell can be a taxol-resistant mammalian cancer cell that overexpresses endogenous TRAG-3 as compared to a non-taxol-resistant cancer cell from a parental cell line. The decrease in the expression level of TRAG-3 gene expression can be detected through measurement of TRAG-3 RNA level in the cell, or through measurement of TRAG-3 polypeptide level in the cell. TRAG-3 polypeptide level can be measured by means of a TRAG-3 antibody.
The invention also includes a screening method for identifying a compound that inhibits the biological activity of a TRAG-3 polypeptide in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which: (1) contain a TRAG-3 coding sequence operably linked to expression control sequences, and (2) express an endogenous gene whose expression is upregulated by a TRAG-3 polypeptide; (b) contacting the treatment test cell with a candidate compound; and (c) detecting a decrease in the expression level of the endogenous gene in the treatment test cell, compared to the expression level of the endogenous gene in the control test cell. The level of TRAG-3 -modulated gene expression can be measured according to TRAG-3 RNA level or TRAG-3 polypeptide level. The endogenous gene whose expression is upregulated by a TRAG-3 polypeptide can be an annexin I gene, IL-6 gene, IL-8 gene, or macrophage inflammatory protein 2xcex1 gene.
The invention also includes a screening method for identifying a compound that inhibits the biological activity of a TRAG-3 polypeptide in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which contain: (1) a TRAG-3 coding sequence operably linked to expression control sequences, and (2) a reporter gene operably linked to a positive expression control sequence naturally associated with an endogenous gene whose expression is upregulated by a TRAG-3 polypeptide; (b) contacting the test cell with a candidate compound; and (c) detecting a decrease in the level of reporter gene expression in the treatment test cell, compared to the level of reporter gene expression in the control test cell.
The invention also includes a screening method for identifying a compound that inhibits the biological activity of a TRAG-3 polypeptide in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which: (1) contain a TRAG-3 coding sequence operably linked to expression control sequences, and (2) express an endogenous gene whose expression is downregulated by a TRAG-3 polypeptide; (b) contacting the treatment test cell with a candidate compound, and (c) detecting an increase in the expression level of the endogenous gene in the treatment test cell, compared to the expression level of the endogenous gene in the control test cell. The endogenous gene whose expression is downregulated by a TRAG-3 polypeptide can be an NKEFB gene.
The invention includes an additional screening method for identifying a compound that inhibits the biological activity of a TRAG-3 polypeptide in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which: (1) contain a TRAG-3 coding sequence operably linked to expression control sequences, and (2) a reporter gene operably linked to a negative expression control sequence naturally associated with an endogenous gene whose expression is downregulated by a TRAG-3 polypeptide; (b) contacting the test cell with a candidate compound; and (c) detecting an increase in the expression level of the reporter gene in the treatment test cell, compared to the expression level of the reporter gene expression in the control test cell.
The invention also includes a method for identifying a cancer tissue suitable for treatment with a compound that inhibits TRAG-3 gene expression or that inhibits the biological activity of a TRAG-3 polypeptide. The method includes: (a) providing a sample of the cancer tissue, and (b) detecting TRAG-3 gene expression in the sample.
The present invention also provides a diagnostic assay for identifying whether a cell is cancerous. The method includes: (a) providing a cell which is suspected of being cancerous and (b) determining whether the cell expresses TRAG-3. Expression of TRAG-3 is an indication that the cell is cancerous. TRAG-3 gene expression can be detected through measurement of TRAG-3 RNA level in the cell, or through measurement of TRAG-3 polypeptide level in the cell.
As used herein, xe2x80x9ca substantially pure TRAG-3 polypeptidexe2x80x9d or xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cisolatedxe2x80x9d means a TRAG-3 polypeptide separated from components that naturally accompany it. Typically, the protein is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure TRAG-3 polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding a TRAG-3 polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically synthesized protein or a recombinant protein produced in a cell type other than the cell type in which it naturally occurs is, by definition, substantially free from components that naturally accompany it. Accordingly, substantially pure proteins include those having sequences derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
As used herein, xe2x80x9chigh stringencyxe2x80x9d means the following nucleic acid hybridization and wash conditions: hybridization at 42xc2x0 C. in the presence of 50% formamide; a first wash at 65xc2x0 C. with 2xc3x97SSC containing 1% SDS; followed by a second wash at 65xc2x0 C. with 0.1xc3x97SSC.
As used herein, xe2x80x9cisolated DNAxe2x80x9d means DNA free of the genes that flank the gene of interest in the genome of the organism in which the gene of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment. It also includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Also included is a recombinant DNA that includes a portion of SEQ ID NO:1 and that encodes an alternative splice variant of RKLF.
As used herein, xe2x80x9coperably linkedxe2x80x9d means incorporated into a genetic construct so that expression control sequences effectively control expression of a gene of interest.
As used herein, xe2x80x9cpolypeptidexe2x80x9d means any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
As used herein, xe2x80x9csequence identityxe2x80x9d means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence of 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. Preferably, the length of the compared sequences is at least 60, more preferably at least 75, and most preferably, at least 100 nucleotides or amino acid residues.
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions include substitutions within the following groups, e.g., glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) with the default parameters as specified therein. BLAST software, provided as a service by the National Center for Biotechnology Information is useful for making sequence comparisons. The programs are described in detail by Karlin et al., (Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 and 90:5873-7, 1993) and Altschul et al., (Nucl. Acids Res. 25:3389-3402, 1997), and they are available on the internet
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned herein are incorporated by reference, in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.